1. Field of the Invention
The present invention relates to a sample information measuring method and a scanning confocal microscope, and more specifically to a sample information measuring method and a scanning confocal microscope for measuring surface information about a height direction of a sample using the scanning confocal microscope, and displaying a three-dimensional shape of the sample in a visually recognizable manner.
2. Description of the Related Art
Conventionally, a scanning confocal microscope applies dotted illumination to a specimen, converges transmitted light, reflected light, or fluorescence from the specimen on a confocal diaphragm, and detects by a photodetector the intensity of the light passing through the confocal diaphragm, thereby obtaining the surface information about the specimen. A scanning confocal microscope scans the surface of the specimen using dotted illumination in various methods, thereby obtain the surface information about the specimen in a wide range.
FIG. 1A shows the outline of the configuration of a conventional scanning confocal microscope.
With the scanning confocal microscope shown in FIG. 1A, a beam output from a light source 1 passes through a beam splitter 2, and enters a two-dimensional scanning mechanism 3. The two-dimensional scanning mechanism 3 has a first optical scanner 3a and a second optical scanner 3b, performs two-dimensional scanning using luminous flux, and leads it to an object lens 7. The luminous flux input to the object lens 7 becomes converging beam and scans the surface of a sample 8.
The light reflected by the surface of the sample 8 is introduced from the object lens 7 again to the beam splitter 2 through the two-dimensional scanning mechanism 3, then reflected by the beam splitter 2, and converges on a pinhole 10 by an image forming lens 9. The pinhole 10 cuts off the reflected light from the points other than the beam condensing point of the sample 8 and a photodetector 11 detects the light only passing through the pinhole 10.
The specimen 8 is held on a sample table 13. A stage 14 and the photodetector 11 are controlled by a computer 12.
The beam condensing position by the object lens 7 is in a position optically conjugate with the pinhole 10. When the sample 8 is in the beam condensing position of the object lens 7, the reflected light from the sample 8 converges on the pinhole 10 and passes through the pinhole 10. When the sample 8 is displaced from the beam condensing position of the object lens 7, the reflected light from the sample 8 does not converges on the pinhole 10, and does not pass through the pinhole 10.
FIG. 1B shows the relationship between the relative position (Z) of the object lens 7 to the specimen 8 and the output (I) of the photodetector 11.
This relation is called I-Z curve as follows.
As shown in FIG. 1B, when the sample 8 is in the beam condensing position Z0 of the object lens 7, the output of the photodetector 11 indicates a maximum value. As the relative position of the object lens 7 to the sample 8 leaves from the position, the output of the photodetector 11 indicates a sudden decrease.
With the characteristic, if the two-dimensional scanning mechanism 3 performs two-dimensional scanning on the beam condensing point, and an image is generated by the output of the photodetector 11 in synchronization with the two-dimensional scanning mechanism 3, then an image of only a specific height portion of the sample 8 is formed, and an image (confocal image) is obtained by optically slicing the sample 8. Furthermore, the sample 8 is discretely moved on the stage 14 in the optical axis direction, the two-dimensional scanning mechanism 3 performs scanning in each position to obtain a confocal image, and the position Z of the stage 14 where the output of the photodetector 11 indicates the maximum value is detected, thereby obtaining the height information about the specimen 8. Additionally, by overlaying and displaying the maximum value of the output of the photodetector 11 at each point of the sample, an image can be obtained with all points of the image displayed in focus (extend image).
When the height of the sample 8 is measured with the above-mentioned configuration, it is necessary to reduce the amount of each travel of the stage 14 to enhance the measurement precision. As a result, it takes some time to make a necessary measurement. Therefore, a height measuring method is proposed to enhance the precision in measuring the height of the sample 8 without reducing each the amount of each travel of the stage 14 (refer to Japanese Patent Laid-open Publication No. Hei 9-68413).
In this method, the output of the photodetector 11 is sequentially obtained while moving the stage 14 based on a predetermined amount of travel. Then, based on the output of the photodetector 11 relating to the three points, that is, the point indicating the maximum value of the output and the points before and after the point indicating the maximum value, an I-Z curve is approximated by a quadratic curve, and the position of the stage 14 where the output of the photodetector 11 is to be the maximum is obtained with the precision equal to or lower than the amount of travel of the stage 14, thereby obtaining the height information.
There is a disclosed technology of obtaining the surface height data H(x,y) as the surface information about the sample corresponding to each pixel based on a confocal image captured at each height in the height direction of the sample with a view to measuring the shape of the surface of the sample with high resolution without reducing the relative amount of travel of the sample in the height direction (refer to Japanese Patent Laid-open Publication No. Hei 9-113235).
Practically, the first height position D(m) where the quantity of light rises to the maximum value in the height direction is obtained, and the first light quantity Fm(x,y) in the first height position D(m) and the second light quantity Fm−1(x,y) and the third light quantity Fm+1(x,y) respectively at the second height position D(m−1) and the third height position D(m+1) respectively close to the upper and lower sides of the first height position D(m) are obtained. Based on these values, a quadratic curve indicating a change of the quantity of light relative to the height position is obtained, and the extreme value of the quality of light is obtained from the quadratic curve. Furthermore, the height position Dmax corresponding to the extreme value is defined as surface height data H(x,y).
Additionally, a scanning confocal microscope capable of obtaining the optical axis direction position and the three-dimensional shape of a sample without scanning in the optical axis direction is disclosed. This scanning confocal microscope includes a laser beam source, a confocal scanner for outputting after passing output light of the laser beam source through an aperture, an optical microscope for converging the output light from the confocal scanner on the sample, a shooting device for shooting the light passing through the aperture of the confocal scanner in the return light from the sample, and obtaining a sectional image, and a control device for obtaining the optical axis direction position of the sample from the quality of light of the sectional image based on the optical axis direction position to light quantity characteristic (Japanese Patent Laid-open Publication No. Hei 11-264933).